Biochemical Aspects of a Serine Protease from Caesalpinia echinata Lam. (Brazilwood) Seeds: A Potential Tool to Access the Mobilization of Seed Storage Proteins

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (K-m 55.7 mu M) in an optimum pH of 7.1, and this activity is effectively retained until 50 degrees C. CeSP remained stable in the presence of kosmotropic anions (PO43-, SO42-, and CH3COO-) or chaotropic cations (K+ and Na+). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.

Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)

(Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)). The cotyledons of Hymenaea courbaril store large amounts of xyloglucan, a cell wall polysaccharide that is believed to serve as storage for the period of seedling establishment. During storage mobilisation, xyloglucan seems to be degraded by a continuous process that starts right after radicle protrusion and follows up to the establishment of photosynthesis. Here we show evidence that events related to the hydrolases activities and production (alpha-xylosidase, beta-galactosidase, beta-glucosidase and xyloglucan endo-beta-transglucosilase) as well as auxin, showed changes that follow the diurnal cycle. The period of higher hydrolases activities was between 6pm and 6am, which is out of phase with photosynthesis. Among the enzymes, alpha-xilosidase seems to be more important than beta-glucosidase and beta-galactosidase in the xyloglucan disassembling mechanism. Likewise, the sugars related with sucrose metabolism followed the rhythm of the hydrolases, but starch levels were shown to be practically constant. A high level of auxin was observed during the night, what is compatible with the hypothesis that this hormone would be one of the regulators of the whole process. The probable biological meaning of the existence of such a complex control mechanism during storage mobilisation is likely to be related to a remarkably high level of efficiency of carbon usage by the growing seedling of Hymenaea courbaril, allowing the establishment of very vigorous seedlings in the tropical forest.

Influence of salinity on the interannual heat storage trends in the Atlantic estimated from altimeters and Pilot Research Moored Array in the Tropical Atlantic data

Changes in the oceanic heat storage (HS) can reveal important evidences of climate variability related to ocean heat fluxes. Specifically, long-term variations in HS are a powerful indicator of climate change as HS represents the balance between the net surface energy flux and the poleward heat transported by the ocean currents. HS is estimated from sea surface height anomaly measured from the altimeters TOPEX/Poseidon and Jason 1 from 1993 to 2006. To characterize and validate the altimeter-based HS in the Atlantic, we used the data from the Pilot Research Moored Array in the Tropical Atlantic (PIRATA) array. Correlations and rms differences are used as statistical figures of merit to compare the HS estimates. The correlations range from 0.50 to 0.87 in the buoys located at the equator and at the southern part of the array. In that region the rms differences range between 0.40 and 0.51 x 10(9) Jm(-2). These results are encouraging and indicate that the altimeter has the precision necessary to capture the interannual trends in HS in the Atlantic. Albeit relatively small, salinity changes can also have an effect on the sea surface height anomaly. To account for this effect, NCEP/GODAS reanalysis data are used to estimate the haline contraction. To understand which dynamical processes are involved in the HS variability, the total signal is decomposed into nonpropagating basin-scale and seasonal (HS(l)) planetary waves, mesoscale eddies, and small-scale residual components. In general, HS(l) is the dominant signal in the tropical region. Results show a warming trend of HS(l) in the past 13 years almost all over the Atlantic basin with the most prominent slopes found at high latitudes. Positive interannual trends are found in the halosteric component at high latitudes of the South Atlantic and near the Labrador Sea. This could be an indication that the salinity anomaly increased in the upper layers during this period. The dynamics of the South Atlantic subtropical gyre could also be subject to low-frequency changes caused by a trend in the halosteric component on each side of the South Atlantic Current.

Spatial competition induces the mobilization of morula cells in the colonial ascidian Didemnum perlucidum (Tunicata: Didemnidae)

The effects of spatial competition among colonial marine organisms are often evident in the contact zones between colonies. These effects are especially pronounced when the interaction results in overgrowth or necrosis of one of the competitors. Ascidians, one of the dominant taxonomic groups in subtidal sessile communities, have specialized morula cells that provide a defense against microbial infections. Injuries resulting from interspecific competitive interactions might also act as a stimulus for this defensive mechanism. Therefore, we expected to see the recruitment of morula cells in tissues near competitor contact zones. To test the hypothesis that spatial competition elicits this immune response, we placed colonies of the ascidian Didemnum perlucidum from southeastern Brazil in four different types of competitive situations: (1) overgrowth of the competitor, (2) stand-off interactions, (3) overgrowth by the competitor, and (4) free of competitors. Our results indicate that competitive interactions increase the population of morula cells in contact zones, as more cells were observed in interactions that resulted in the overgrowth of individuals of D. perlucidum, and fewer cells were observed in colonies that were free of competitors. We identified the defensive function of the morula cells by showing the presence of the enzyme phenoloxidase within its vacuoles. Phenoloxidase is a widespread enzyme among animals and plants, and is frequently used in defense by synthesizing toxic quinones from polyphenol substrates. This is the first study to document the presence of morula cells in didemnid ascidians and the mobilization of these cells by spatial competition by heterospecifics, and one of the first studies to identify phenoloxidase activity in morula cells.

Distribution and stability of aflatoxin M-1 during processing and storage of Minas Frescal cheese

Aflatoxin M-1 (AFM(1)) is a hepatocarcinogen found in milk of animals that have consumed feeds with aflatoxin B-1. The carry-over of AFM(1) from milk to Minas Frescal cheese produced with or without starter cultures was determined. 40 L of milk were divided into 10 L each and assigned to the following treatments for cheese manufacture: 0.250 rig AFM(1) mL(-1), 0.500 rig AFM(1) mL(-1), 0.250 ng AFM(1) mL(-1) + starter, 0.500 ng AFM(1) mL(-1) + starter. Quantification of AFM(1) was achieved by high performance liquid chromatography. The carry-over of AFM(1) from milk to cheese ranged from 30.64% to 42.26%. There was no effect of storage time on AFM(1). Milk with AFM(1) in levels studied may concentrate the toxin in Minas Frescal cheese, but at concentrations below the Brazilian tolerance limit. The addition of starter cultures did not influence concentration or stability of the AFM(1) in cheese over 30 days storage. (C) 2011 Elsevier Ltd. All rights reserved.

Influence of different banana cultivars on volatile compounds during ripening in cold storage

The aroma responsible for the flavor of fruits is highly susceptible to low temperatures in storage. The present study investigated the volatile composition of the Nanicao and Prata banana cultivars by testing pulp and whole fruit under cold storage conditions. The volatile fractions were characterized using headspace solid phase micro-extraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The cold storage induced changes in the volatile profile relative to the profile of the control group. The result of principal component analysis revealed that cold storage more strongly affects the Nanicao than the Prata cultivar. Esters such as 2-pentanol acetate, 3-methyl-1-butanol acetate, 2-methylpropyl butanoate, 3-methylbutyl butanoate, 2-methylpropyl 3-methylbutanoate and butyl butanoate were drastically reduced in the cold group of the Nanicao cultivar. Our results suggest that the metabolism responsible for the production of volatile compounds is related to the ability to tolerate low temperatures. (C) 2012 Elsevier Ltd. All rights reserved.

Effect of sample storage time on detection of hybridization signals in Checkerboard DNA-DNA hybridization

Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the hybridization signals of 13 bacterial species detected by the Checkerboard DNA-DNA hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n = 24) and (2) after storage at -20 degrees C for 6 months (n = 24), (3) for 12 months (n = 24), and (4) for 24 months (n = 24). The intensity of hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p < 0.001). No differences were found between groups 1 and 2 (p > 0.05). The Checkerboard DNA-DNA hybridization method was suitable to detect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periods of sample storage.

Effects of curing protocol and storage time on the micro-hardness of resin cements used to lute fiber-reinforced resin posts

Objectives: To determine the micro-hardness profile of two dual cure resin cements (RelyX - U100 (R), 3M-ESPE and Panavia F 2.0 (R), Kuraray) used for cementing fiber-reinforced resin posts (Fibrekor (R) - Jeneric Pentron) under three different curing protocols and two water storage times. Material and methods: Sixty 16mm long bovine incisor roots were endodontically treated and prepared for cementation of the Fibrekor posts. The cements were mixed as instructed, dispensed in the canal, the posts were seated and the curing performed as follows: a) no light activation; b) light-activation immediately after seating the post, and; c) light-activation delayed 5 minutes after seating the post. The teeth were stored in water and retrieved for analysis after 7 days and 3 months. The roots were longitudinally sectioned and the microhardness was determined at the cervical, middle and apical regions along the cement line. The data was analyzed by the three-way ANOVA test (curing mode, storage time and thirds) for each cement. The Tukey test was used for the post-hoc analysis. Results: Light-activation resulted in a significant increase in the microhardness. This was more evident for the cervical region and for the Panavia cement. Storage in water for 3 months caused a reduction of the micro-hardness for both cements. The U100 cement showed less variation in the micro-hardness regardless of the curing protocol and storage time. Conclusions: The micro-hardness of the cements was affected by the curing and storage variables and were material-dependent.

Neural mobilization reverses behavioral and cellular changes that characterize neuropathic pain in rats

Background: The neural mobilization technique is a noninvasive method that has proved clinically effective in reducing pain sensitivity and consequently in improving quality of life after neuropathic pain. The present study examined the effects of neural mobilization (NM) on pain sensitivity induced by chronic constriction injury (CCI) in rats. The CCI was performed on adult male rats, submitted thereafter to 10 sessions of NM, each other day, starting 14 days after the CCI injury. Over the treatment period, animals were evaluated for nociception using behavioral tests, such as tests for allodynia and thermal and mechanical hyperalgesia. At the end of the sessions, the dorsal root ganglion (DRG) and spinal cord were analyzed using immunohistochemistry and Western blot assays for neural growth factor (NGF) and glial fibrillary acidic protein (GFAP). Results: The NM treatment induced an early reduction (from the second session) of the hyperalgesia and allodynia in CCI-injured rats, which persisted until the end of the treatment. On the other hand, only after the 4th session we observed a blockade of thermal sensitivity. Regarding cellular changes, we observed a decrease of GFAP and NGF expression after NM in the ipsilateral DRG (68% and 111%, respectively) and the decrease of only GFAP expression after NM in the lumbar spinal cord (L3-L6) (108%). Conclusions: These data provide evidence that NM treatment reverses pain symptoms in CCI-injured rats and suggest the involvement of glial cells and NGF in such an effect.

Improved low-voltage-ride-through capability of fixed-speed wind turbines using decentralised control of STATCOM with energy storage system

The design and implementation of a new control scheme for reactive power compensation, voltage regulation and transient stability enhancement for wind turbines equipped with fixed-speed induction generators (IGs) in large interconnected power systems is presented in this study. The low-voltage-ride-through (LVRT) capability is provided by extending the range of the operation of the controlled system to include typical post-fault conditions. A systematic procedure is proposed to design decentralised multi-variable controllers for large interconnected power systems using the linear quadratic (LQ) output-feedback control design method and the controller design procedure is formulated as an optimisation problem involving rank-constrained linear matrix inequality (LMI). In this study, it is shown that a static synchronous compensator (STATCOM) with energy storage system (ESS), controlled via robust control technique, is an effective device for improving the LVRT capability of fixed-speed wind turbines.

Experimental Adhesives with Different Hydrophilicity: Microshear Test in after 1, 7, and 90 Days' Storage

Purpose: To assess the microshear bond strength of 3 experimental adhesives with different degrees of hydrophilicity after 1, 7 and 90 days of storage. Materials and Methods: The bonding effectiveness of three experimental two-step etch-and-rinse adhesives (bis-GMA, bis-EMA/bis-GMA, polybutadiene [C6H12]) and one commercial adhesive (Single Bond) to sound hydrated dentin was determined using the nnicroshear test with delimitation of the adhesive area after 1, 7, and 90 days of storage in water at 37 degrees C. Two-way ANOVA was performed at the 0.05 probability level. The fractures were classified as adhesive, cohesive in dentin, cohesive in resin, and mixed. Results: The experimental adhesives showed values in the range of 11.31 to 12.96 MPa, with polybutadiene (PBH) showing the lowest bond strengths, bis-GMA the highest, and bis-EMA/bis-GMA intermediary values. Single Bond yielded bond strengths of approximately 24 MPa. Water storage decreased the bond strength in all adhesives. Adhesive fractures were predominant in experimental adhesives, while mixed fractures were the most frequent type in the Single Bond group. Conclusion: The experimental dentin adhesives of this study were able to form resin tags, but they could not penetrate into the collagen fibers and form hybrid layers. The resulting low bond strength decreased with increasing length of storage.

VALIDATION OF THE LAND WATER STORAGE FROM GRAVITY RECOVERY AND CLIMATE EXPERIMENT (GRACE) WITH GAUGE DATA IN THE AMAZON BASIN

The Amazon basin is a region of constant scientific interest due to its environmental importance and its biodiversity and climate on a global scale. The seasonal variations in water volume are one of the examples of topics studied nowadays. In general, the variations in river levels depend primarily on the climate and physics characteristics of the corresponding basins. The main factor which influences the water level in the Amazon Basin is the intensive rainfall over this region as a consequence of the humidity of the tropical climate. Unfortunately, the Amazon basin is an area with lack of water level information due to difficulties in access for local operations. The purpose of this study is to compare and evaluate the Equivalent Water Height (Ewh) from GRACE (Gravity Recovery And Climate Experiment) mission, to study the connection between water loading and vertical variations of the crust due to the hydrologic. In order to achieve this goal, the Ewh is compared with in-situ information from limnimeter. For the analysis it was computed the correlation coefficients, phase and amplitude of GRACE Ewh solutions and in-situ data, as well as the timing of periods of drought in different parts of the basin. The results indicated that vertical variations of the lithosphere due to water mass loading could reach 7 to 5 cm per year, in the sedimentary and flooded areas of the region, where water level variations can reach 10 to 8 m.

Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT(1)/AT(2) receptors to activate SERCA and to promote Ca2+ mobilization

Ferrao FM, Lara LS, Axelband F, Dias J, Carmona AK, Reis RI, Costa-Neto CM, Vieyra A, Lowe J. Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT(1)/AT(2) receptors to activate SERCA and to promote Ca2+ mobilization. Am J Physiol Renal Physiol 302: F875-F883, 2012. First published January 4, 2012; doi:10.1152/ajprenal.00381.2011.-ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK1 cells on Ca2+-ATPase of the sarco(endo) plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca2+ mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca2+ spark, demonstrating a positively self-sustained stimulus of Ca2+ mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC -> DAG(PMA)-> PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca2+ stock in the reticulum to ensure a more efficient subsequent mobilization of Ca2+. This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca2+ homeostasis in renal cells and also for regulation of Ca2+-modulated fluid reabsorption in proximal tubules.

Development of an Autonomous Robot for Gas Storage Spheres Inspection

Inspection for corrosion of gas storage spheres at the welding seam lines must be done periodically. Until now this inspection is being done manually and has a high cost associated to it and a high risk of inspection personel injuries. The Brazilian Petroleum Company, Petrobras, is seeking cost reduction and personel safety by the use of autonomous robot technology. This paper presents the development of a robot capable of autonomously follow a welding line and transporting corrosion measurement sensors. The robot uses a pair of sensors each composed of a laser source and a video camera that allows the estimation of the center of the welding line. The mechanical robot uses four magnetic wheels to adhere to the sphere's surface and was constructed in a way that always three wheels are in contact with the sphere's metallic surface which guarantees enough magnetic atraction to hold the robot in the sphere's surface all the time. Additionally, an independently actuated table for attaching the corrosion inspection sensors was included for small position corrections. Tests were conducted at the laboratory and in a real sphere showing the validity of the proposed approach and implementation.

The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle

The in vitro stability of cocaine in horse blood, sheep vitreous humour (VH) and homogenised deer muscle is described. The stability of cocaine in horse blood was of interest because many toxicology laboratories utilise horse blood for the preparation of calibration and check standards and the latter are typically stored during routine use. The storage stability of cocaine in human VH and muscle has not been previously reported. In the absence of blank human VH and muscle, cocaine stability under varying conditions was demonstrated in animal tissues. Blood and VH were stored with and without addition of NaF at room temperature (RT), 4 degrees C and -18 degrees C for 84 days. Muscle homogenates were prepared in water, water/2% NaF, and phosphate buffer (pH 6.0)/2% NaF, and stored for 31 days at RT, 4 degrees C and -18 degrees C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18 degrees C for 13 months. Cocaine and benzoylecgonine (BZE) were extracted using SPE and quantified by GC-MS/MS. Cocaine was stable for 7 days in refrigerated (4 degrees C) horse blood fortified with 1 and 2% NaF. In the absence of NaF, cocaine was not detectable by day 7 in blood stored at RT and 4 degrees C and had declined by 81% following storage at -18 degrees C. At 4 degrees C the rate of cocaine degradation in blood preserved with 2% NaF was significantly slower than with 1% NaF. The stability of cocaine in horse blood appeared to be less than that reported for human blood, probably attributable to the presence of carboxylesterase in horse plasma. Cocaine stored in VH at -18 degrees C was essentially stable for the study period whereas at 4 degrees C concentrations decreased by >50% in preserved and unpreserved VH stored for longer than 14 days. Fluoride did not significantly affect cocaine stability in VH. The stability of cocaine in muscle tissue homogenates significantly exceeded that in blood and VH at every temperature. In preserved and unpreserved samples stored at 4 degrees C and below, cocaine loss did not exceed 2%. The increased stability of cocaine in muscle was attributed to the low initial pH of post-mortem muscle. In tissue from one human case stored for 13 months at -18 degrees C the muscle cocaine concentration declined by only 15% (range: 5-22%). These findings promote the use of human muscle as a toxicological specimen in which cocaine may be detected for longer compared with blood or VH. (C) 2011 Elsevier Ireland Ltd. All rights reserved.